Methods and compositions for the direct concentrated delivery of passive immunity

ABSTRACT

Compositions containing a high concentration of the full repertoire of immunoglobulins, including IgA, IgM and IgG, are used to combat infections from microorganisms and viruses at a wound, surgical, or burn site, or normal tissue at times of risk of infection. The compositions can contain elevated antibody titers for several specific pathogens including S. aureus, CNS, Enterococci, S. epidermidis, P. aeruginosa, E. coli, and Enterobacter spp., etc. The compositions are applied directly to a wound or burn site as an ointment, creme, fluid, spray, or the like, prior to viral or bacterial attachment or biofilm formation such that adhesion of the pathogens is inhibited and the pathogens closest to the wound or burn site will be pre-opsonized for phagocytic killing prior to toxin release. The immunoglobulins in the composition can be immobilized on a biocompatible material such as collagen, fibrin, hyaluronan, biodegradable polymers, and fragments thereof, which will be placed in-situ at the wound, surgical or burn site. In addition, the immunoglobulins in the composition may be coated on the body contacting surface of an implantable device such as a catheter, contact lens or total joint. The inventive compositions have particular application in preventing infections.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of application Ser. No. 08/295,482filed Aug. 25, 1994, which itself is a continuation application of Ser.No. 08/003,305 filed Jan. 12, 1993, now abandoned, which is hereinincorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is directed to the in situ precoating and preopsonizationby direct application of a full repertoire of immunoglobulins (IgG, IgA,IgM, and parts thereof) to the surfaces of tissues and biomaterials forthe prevention and treatment of microbial adhesion, colonization, andinfection in man and animals.

2. Description of the Prior Art

Surgical wound infection, especially biomaterial centered, or sepsissubsequent to major trauma with bacterial contamination, continues to bea significant problem in morbidity and cost even with the use ofantibiotics. There are twenty five million surgeries each year in theUnited States and an equivalent number in Europe. Although rates ofinfection are quite low for most elective surgeries, they aresignificantly higher in the presence of biomaterial implants ortraumatized tissue and range from less than 1% for total hips, to 6% forvascular grafts, half of which culminate in amputation or death. Therate of infection for the total artificial heart is essentially 100% ifawaiting bridge to transplant for more than ninety days. Most often, andinterestingly, infections about biomaterials cannot be cured withoutremoval of the implant, even with massive doses of antibiotics. Majorcontaminated wounds and open fractures such as occur in industry, autotrauma, and warfare also have up to and more than a 10% rate of sepsis.Biomaterial centered infection is discussed in Gristina, Science,237:1588-1595 (1987), Gristina et al., JAMA, 259:870-874 (1988), andGristina et al., "Molecular Mechanisms in Musculoskeletal Sepsis: TheRace for the Surface", Chapter 58 of Instructional Course Lectures, Vol.XXXIX 1990, ed. Greene, American Academy of Orthopaedic Surgeons.

All burns are colonized by bacteria. Large 2° and 3° burns producesevere local and systemic sepsis, toxin release, additional tissuedestruction, and bacteremia.

Streptococcal infections, endocarditis, and pneumonia also persist asserious problems for at risk groups. Tuberculosis and secondaryopportunistic pathogens are among the recurring diseases in immunocompromised patients (AIDs). For these diseases, antibiotics are oftenineffective, not timely or deliverable. Respiratory, genitourinary, andgynecologic mucosal surfaces are vulnerable to recurrent and chronicbacterial and viral invasion.

The two important causal mechanisms for these infections are: (1)microbial adhesion to damaged tissue or biomaterial substrata and theformation of bacterial biofilms which shield microorganisms from hostdefenses and antibiotics, and (2) disruption of host defenses and theproduction of an immunoincompetent inflammatory zone at damaged tissuesand biomaterial interfaces. Biomaterial surfaces, their particulatedebris, severe tissue trauma, and burns cause massive and chronicinflammatory responses characterized by host defense mechanismexhaustion. Additionally 1° and 2° immuno deficiency states (e.g., AIDs,the aged, diabetics, etc.) cause increased host susceptability topathogens.

Currently antibiotics are the treatment of choice for most bacterialdiseases, but they tend to be ineffective against contaminated openfracture, biomaterial centered, foreign body and burn infections, cannotbe extensively used to preempt infection, and do not potentiate hostdefenses. Antibiotics and host defenses (immunoglobulins) usually areineffective after bacteria have formed protective biofilms (see,Gristina, Science, 237:1588-1595 (1987), Gristina et al., JAMA,259:870-874 (1988), and Gristina et al., "Molecular Mechanisms inMusculoskeletal Sepsis: The Race for the Surface", Chapter 58 ofInstructional Course Lectures, Vol. XXXIX 1990, ed. Greene, AmericanAcademy of Orthopaedic Surgeons). Furthermore, use of antibiotics causesselection for the survival of drug-resistant strains.

Higher animals have, by evolution, established several very effectivemeans of defense against microbes involving the immune system. Invadingbacteria are rapidly identified, via complement and immunoglobulinopsonization, phagocytized and destroyed by the cellular immune systemand white blood cells (neutraphils and macrophages). Globulins areessentially nature's perfect antibodies. Complement, available as aprecursor protein which is activated by the presence of microorganismsand globulins, also functions in antibacterial activities. Opsonizationof foreign organisms is the memory component of the immune system. Afterprevious antigenic exposure, the immune system produces a series ofglobulins which attach to and coat bacteria or neutralize viruses sothat they are readily recognized, phagocytosed and destroyed byneutrophils and macrophages. Foreign proteins of invading organisms alsostimulate a humoral immune response which over a period of time (3-6weeks) amplifies the numbers of cells designed to recognize and destroyspecific invaders. Tables 1 and 2 present the antimicrobial functions ofimmunoglobulins and the metabolic properties of immunoglobulins.

TABLE 1

Antimicrobial functions:

(1) Bacterial lysis (requires complement)

(2) Opsonization (enhanced by complement)

(3) Toxin neutralization

(4) Viral neutralization (may be enhanced by complement)

(5) Mediates antibody dependent cell mediated cytoxicity (ADCC)

(6) Synergistic activity with antibiotics

                  TABLE 2                                                         ______________________________________                                        Metabolic Properties of Immunoglobulins                                               IgG     IgA      IgM      IgD  IgE                                    ______________________________________                                        Serum Level                                                                             989       200      100    3    0.008                                Mean (mg/dl)                                                                  (range)   (600-1600)                                                                              (60-330) (45-150)                                         Total Body                                                                              1030      210      36     1.1  0.01                                 Pool mean                                                                     (mg/kg)                                                                       (range)   (570-2050)                                                          Synthesis rate                                                                          36        28       2.2    0.4  0.004                                mean                                                                          (mg/kg/day)                                                                   Plasma half life                                                                        21        5.9      5.1    2.8  2.4                                  mean (days)                                                                   Fractional turn-                                                                        6.9       24.0     10.6   37.0 72.0                                 over rate                                                                     (% day) mean                                                                  Fraction for                                                                            0.52      0.55     0.74   0.75 0.51                                 each class in                                                                 plasma.sup.a mean                                                             ______________________________________                                         .sup.a This fraction represents the portion of the total immunoglobulins      of each class that is found in the plasma.                               

Host responses are initiated only after bacteria or viruses have alreadycolonized tissues or implants and are beginning to enhance their owndefenses (antigen masking, replication, biofilm, toxins). The hostdefense strategies require time to reach peak responses. During thistime period, serious infection may be established, especially inimmuno-compromised patients. The presence of tissue damage and foreignbodies lower thresholds of infection and diminishes effective responses.

In the last decade, intravenous immunoglobulins (IVIG) have become amajor treatment regime for bacterial and viral infections and of primaryand secondary immunodeficiency states. For example, Buckley et al., NewEng. J. Med. 325:110-117 (1991), describe using intravenous immuneglobulin in the treatment of immunodeficiency diseases, and Cometta etal., New Eng. J. Med. 327:234-239 (1992), describe the prophylacticintravenous administration of standard immune globulin andcore-lipopolysaccharide immune globulin in patients at high risk ofpost-surgical infection. IVIGs are prepared from the pooled plasmas oflarge numbers of donors, and tend to have a broad representation ofantibodies. Specifically, pooled polyvalent human globulins usuallycontain antibodies for ubiquitous pathogens such as H. influenza type b,pneumococci, staphylococci, diphtheria, tetanus, respiratory synctialvirus (RSV), measles, cytomegalovirus (CMV), and varicella zoster virus.Antibody concentrations from lot to lot and from manufacturer tomanufacturer usually vary only two to four fold when measured byantibody binding assays. However, functional assays often show muchlarger lot to lot variations as do antibody concentrations to lesscommon pathogens (see, Siber et al., "Use of immune globulins in theprevention and treatment of infections", Current Clinical Topics inInfectious Disease, Remington JS, Swartz MM, eds., Blackwell Scientific,Boston, 12:208- 257 (1992)).

IVIG therapy has been reported to be beneficial for more than thirtyfive diseases produced by immunopathologic mechanisms. Passiveimmunization against infections has been particularly successful withimmune globulins specific for tetanus, hepatitis B, rabies, chickenpox,and cytomegalovirus. Passive immunization depends on the presence ofhigh and consistent titers of antibodies to the respective pathogens ineach preparation.

Nosocomial infections are derived from the hospital or clinical setting,and are also a serious problem. Specifically, bacteria and virusespresent in the hospital or clinic can infect a recovering patient andput the patient at risk or prolong the recovery period. A patient's riskfactors for nosocomial infection can be intrinsic, such assusceptibility to infection due to immunosuppression, or extrinsic, suchas invasive medical interventions (e.g., surgery or use of medicaldevices such as catheters, ventilators, etc.). Staphylococcus aureus isan important cause of nosocomial infection, especially nosocomialpneumonia, surgical wound infection, and bloodstream infection (Panlilioet al., Infect. Cont. Hosp. Epidemiol. 13:582-586 (1992)). Otherpathogens commonly associated with nosocomial infection include, but arenot limited to, Escherichia coli, Pseudomonas aeruginosa, Enterococcusspp., Enterobacter spp., coagulase-negative staphylococci (CNS), andCandida albicans (Emori et al., Am. J. Med. 91: (suppl 3B) 289S-293S(1991)). Hospitals and clinics typically employ strict sterilizationprocedures and use antibiotics such as methicillin, oxacillin, andnafcillin to combat virulent bacterial pathogens. However, nosocomialinfections still occur in great numbers and are expected to increasewith an aging population.

The use of intravenous immunoglobulins to prevent nosocomial infectionshas been discussed in Siber, New Eng. J. Med. 327:269-271 (1992).Passive immunization against infections has been particularly successfulusing immune globulins containing antibodies specific for tetanus,hepatitis B, rabies, chickenpox, and CMV. However, it is reported thatthere is an inconsistent benefit from using intravenous immune globulinsto prevent nosocomial infections. This may be due to variable lot-to-lotlevels of antibodies to the more common nosocomial pathogens andemerging new serotypes.

U.S. Pat. No. 4,412,990 to Lundblad et al. discloses an intravenouspharmaceutical composition containing immunoglobulin (IgG) andfibronectin that exhibits a synergistic opsonic activity which resultsin enhanced phagocytosis of bacteria, immune complexes, and viruses.

U.S. Pat. No. 4,994,269 to Collins et al. discloses the topical use ofmonoclonal antibodies for the prevention and treatment of experimentalP. aeruginosa lung infections. Specifically, the antibodies areadministered via aerosol spray to the lungs. Results show beneficialeffects in the treatment of Pseudomonas pneumonia.

U.S. Pat. No. 4,714,612 to Nakamura et al. discloses the use of anon-specific gamma globulin IgG in a mouthwash for preventinggingivitis. Ma et al., Arch. Oral Biol., 35 suppl:115S-122S, 1990,discloses the use of monoclonal antibodies specific for Streptococcusmutans in a mouthwash. Experiments showed control subjects experiencedrecolonization with Streptococcus mutans within two days, but thosetreated with the monoclonal antibodies remained free of Streptococcusmutans for up to two years.

SUMMARY OF THE INVENTION

It is an object of this invention to provide a new method for thedirect, concentrated local delivery of passive immunity.

It is another object of this invention to provide new compositions whichinclude a full repertoire of immunoglobulin classes (IgG, IgA, IgM), andnew methods for prophylactic positioning of the compositions wherein thecompositions are applied directly to wounds, burns, tissues, andbiomaterial devices as a creme, ointment, coating, layer, or the like,to prevent and treat infection from microorganisms and viruses.

It is another object of this invention to provide new compositions,which can include a full repertoire of immunoglobulin classes (IgG, IgA,IgM), and has a broad spectrum of antibodies with elevated antibodytiters to specific microorganisms that commonly cause biomaterial, burn,mucosal, tissue, surgical wound, and body cavity infections.

It is another object of this invention to provide a biocompatible layerwith an immunoglobulin composition containing a broad spectrum ofantibodies to specific infectious pathogens immobilized thereon that isplaced in-situ in the treatment of wounds and burns.

It is another object of this invention to coat catheters and the like,which are used for acute or chronic treatment, with a compositioncontaining a broad spectrum of immunoglobulins which includes antibodiesto prevent the types of infections which often result with the long termuse of these devices.

It is another object of this invention to provide a method of usingimmunoglobulin compositions of broad spectrum and high concentration,whereby bacteria are pre-opsonized in-situ for enhanced phagocytosis andkilling.

According to the invention, the direct, concentrated local delivery ofpassive immunity is accomplished by applying a composition having a fullrepertoire of immunoglobulins (IgG, IgM and IgA) to biomaterials,implants, tissues, and wound and burn sites. The composition preferablyhas elevated concentrations of certain immunoglobulin classes (IgG, IgM,and IgA), and elevated antibody titers to specific microorganisms thatcommonly cause biomaterial, burn, mucosal, tissue, surgical wound, andbody cavity infections. Compositions within the practice of thisinvention may take several forms, including cremes, ointments, lavagefluids, sprays, lozenges, coatings, layers, or any other topical mode ofadministration. In addition, the compositions may be combined with orimmobilized on a biocompatible or biodegradable material, or beimpregnated in a matrix material for sustained release. The compositionscan be used for both prevention and treatment of infections.

In oral applications, the composition would ideally be provided as alozenge, mouthwash, or spray, while in trauma patients the compositionmay be best applied as a creme or ointment, or as part of a biomaterialimplant or fixation device. The immunoglobulins and other antibodies ofthe present compositions can be immobilized on a biocompatible materialwhich is placed in-situ in a patient's wound or burn site, or be coatedon a catheter or the like that is inserted in a body cavity.

Application of the compositions should occur within six hours or at atime of trauma or of cleaning the wound or burn site so that bacteriapresent therein or arriving at the site will be pre-opsonized forphagocytosis and killing prior to their replication and potential toxinproduction. Furthermore, application prior to biofilm formation reducesthe adhesion of infectious bacteria to biomaterial implants and certaintissues, and helps prevent the formation of a biofilm which would blockcontact of the infectious bacteria with circulating immunoglobulins andmacrophages.

In summary, tissue, wound or biomaterial surface pretreatment at thetime of surgery or shortly after trauma, would allow the effective useof a full repertoire of immunoglobulins, including IgG, IgM, and IgA athigh concentrations without side effects, before colonization andinfection develops.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

It is well established that the microorganisms that are causative agentsof biomaterial-related infections have a strong affinity for binding tothe surfaces of biomaterials (See, Gristina et al., "Materials, Microbesand Man: The Problem of Infection Associated with Implantable Devices",Current Perspectives on Implantable Devices, Vol. 1, pp. 71-137 (1989),JAI Press, Inc.). This affinity allows these causative agents of seriousbiomaterial related infections to colonize the surfaces of biomaterials.At the moment of implantation, a polymeric biomaterial, such as avascular graft or the like, is a ready site for competitive bacterial ortissue colonization. In vivo, available bacteria may defeat the hosttissue cells in a race for the polymer's surface and thus causeinfection, resulting in the failure of tissue integration, of thepolymer (Gristina et al., Zbl. Bakt. Suppl. 16, Gustav Fischer Verlag,Stuttgart, New York, pp. 143-157 (1987)). Bacteria colonized on thesurface of a biomaterial become protected from antibiotics and hostdefenses (immunoglobulins) by a biofilm and continuously maintain theinfection in the patient, despite antibiotic medication. The biofilmalso provides the bacteria with some protection from phagocytes, a majormechanism of host defense. Experience has shown that phagocytes havegreat difficulty in their attempts to phagocytose and kill the offendingorganisms growing at the biomaterial-host tissue interface, particularlywhen bacteria are embedded in a biofilm.

Experiments have shown that hyperimmune sera made in rabbits byinjecting rabbits with killed Staphylococcus epidermidis (RP12 strain)and/or the polysaccharide capsular slime extracted from S. epidermidisstrain RP12 markedly reduces the adherence of the RP12 strain to thesurface of the biomaterial polymethylmethacrylate (PMMA). S.epidermidis, which is usually thought of as a nonpathogenic commensalhuman skin saprophyte, has emerged as a serious pathogen inbiomaterial-related infections as well as in immunocompromised patients(Gristina et al., Zbl. Bakt. Suppl. 16, Gustav Fischer Verlag,Stuttgart, New York, pp. 143-157 (1987)). In these experiments, standardsuspensions of the RP12 strain of S. epidermidis were incubated forthirty minutes with 1:200 dilutions of either normal rabbit serum orhyperimmune serum against the RP12 strain of S. epidermidis. Thisallowed the specific antibodies to bind to the surface polysaccharidemolecules of the organisms. These suspensions were washed with phosphatebuffered saline (PBS) and standard samples of PMMA were added to thevarious preparations. The bacteria-PMMA preparations were incubated forsixty minutes, and the PMMA samples were then washed three times withPBS to remove loosely attached bacteria. The PMMA samples were sonicatedfor ten minutes in PBS and the supernatants were diluted and plated onTrypticase-Soy agar to determine the number of colony forming units(CFU) that adhered to the PMMA samples. Table 3 presents theexperimental results.

                  TABLE 3                                                         ______________________________________                                        Effect of Anti-RP12 Antisera on the Binding of the                            RP12 Strain of S. epidermidis to PMMA                                         PMMA plus RP12     CFU Bound  Percent                                         incubated with     to PMMA    inhibition                                      ______________________________________                                        PBS                393,000                                                    Normal Serum (1:200)                                                                             319,000                                                    Antiserum (1:200; lot 11949)                                                                     105,000    67.sup.a 73.sup.b                               ______________________________________                                         .sup.a Calculated as the percent inhibition of antisera treated RP12          versus RP12 pretreated with normal sera.                                      .sup.b Calculated as the percent inhibition of antisera-treated RP12          versus RP12 pretreated with only PBS.                                    

Table 3 shows that normal serum has some inhibitory effects. This is notsurprising because a low level of antibody would be expected in the seraof rabbits and humans because S. epidermidis is a normal floramicroorganism of the skin.

The immunoglobulin G (IgG) fraction was isolated from the antiserum(11949) and tested for its capacity to block adherence of the RP12strain. Gristina, Science 237:1588-1595 (1987), points out thatinhibiting bacterial adhesion is an important parameter in reducingbiomaterial-centered infection. Polymethylmethacrylate (PMMA) samplesincubated with RP12 suspended in PBS (no antibodies) bound 604,000 CFUper sample. In sharp contrast, PMMA samples incubated with RP12preincubated with the hypermimmune IgG only bound 33,000 organisms. Thisrepresents a 94 percent inhibition of binding of RP12 to PMMA.

Kojima et al., J. Infectious Dis. 162:435-441 (1990), disclose similarexperiments where antibody to the capsular polysacharide/adhesin (PS/A)protects rabbits against catheter related bacteremia due to coagulasenegative staphylococci. In vitro experiments with antibody raised toPS/A inhibited adherence of homologous and heterologous adhesin-positivecoagulase negative staphylococci to silicon elastomer catheter tubing ina dose-response fashion.

For comparison purposes, experiments were conducted to determine thecapacity of antiserum (11949) to inhibit the binding of various strainsof coagulase negative staphylococci. Six strains of coagulase negativestaphylococci were incubated with the anti-RP12 antiserum (11949) todetermine whether specificity exists with respect to blocking theadherence of the different strains to PMMA. The inhibition assaydescribed above was performed for each strain and the results are setforth in Table 4.

                  TABLE 4                                                         ______________________________________                                        Capacity of Anti-RP12 Antiserum to Block Adherence                            of Six Strains of Coagulase Negative Staphylococci                            to PMMA                                                                       Strain       CFU/Sample % Inhibition                                          ______________________________________                                        RP12         198,000-6,000                                                                            67-99                                                 SP2          162,000    73                                                    SE360        602,000    0                                                     LD1          126,000    79                                                    ERI          610,000    0                                                     RP62A        695,000    0                                                     ______________________________________                                    

The results in Table 4 indicate that there is specificity in inhibitionand that serologic groups of adhesins exist.

From the above data in Tables 3 and 4, this invention contemplates thathyperimmune sera raised against a pool of adhesins is needed to blockadherence of various coagulase negative serotypes of staphylococci andother bacteria and viruses to biomaterials and to lower the risk ofinfection at surgery.

IVIG compositions are commercially available (e.g., GAMMAGARD® availablefrom Baxter Healthcare Corporation), and are used in the treatment ofprimary immunodeficiency states such as congenital agammaglobulinemias,common variable immunodeficiency, Wiskott-Aldrich syndrome, etc. Inaddition, IVIG compositions have been used to prevent bacterialinfections in patients with hypogammaglobulinemia and/or recurrentbacterial infections associated with B-cell Chronic LymphocyticLeukemia. Siber et al., "Use of immune globulins in the prevention andtreatment of infections", Remington I. S. and Swartz M. N. eds., CurrentClinical Topics in Infectious Disease, Vol. 12, Blackwell Scientific,pp. 203-257, 1992, provides a thorough review of the use of intravenousimmunoglobulins.

The major benefit of the intravenous immunoglobulins may be to opsonizebacteria for phagocytosis and enhance clearance of bacteria or theirproducts. Additional benefits may be the neutralization of endotoxinsand exotoxins, down regulation of interleukin-1 (IL-1) and TNFresponses, and the recruitment of neutrophils from storage pools via C3and C5 fragments. However, intravenous immunoglobulins can havedetrimental effects, including the generation of immune complexesbetween exogenous antibody and large amounts of microbial antigens withthe ensuing release of inflammatory mediators, blockade of Fc receptorsor of C3 fixation leading to impaired clearance mechanisms, enhancedrelease of toxic products such as endotoxin or bacterial cell wallconstituents due to complement mediated bacterial lysis. Experimentswith rats have shown that high doses of intravenous immunoglobulins haveenhanced mortality rates.

In addition, Siber et al., "Use of immune globulins in the preventionand treatment of infections", Remington I. S. and Swartz M. N. eds.,Current Clinical Topics in Infectious Disease, Vol. 12, BlackwellScientific, pp. 203-257, 1992., report that the administration ofintravenous immunoglobulins did not reduce the incidence of bacteremiasor mortality in trauma, major surgery, and burn victims.

This invention is particularly directed to new broad spectrumimmunoglobulin compositions with a full repertoire of immunoglobulinclasses (IgG, IgA, IgM) which are used to prevent and treat infectionsassociated with major surgery, burns, trauma, and biomaterial devicesand implants. In contrast to IVIG compositions, the compositions of thepresent invention are applied directly to the wound or burn site, or thebiocompatible device or implant (including metal and polymericmaterials).

It is probable that intravenous delivery routes cause serum dilution sothat only low concentrations of antibodies reach the specific targetsite. The formation of biofilm protected infections, and limiteddiffusion, at traumatized tissue sites or in sites of poor vascularity(musculoskeletal and joints, burn sites) is also a likely cause for theineffectiveness of IVIGs noted by Siber et al. against trauma. Majortrauma also increases catabolic effects which may alter half-life ofIVIG preparations. The use of IVIG in established infection aftermicroorganisms have adhered, produced toxins, or are intracellular, isalso less likely to be effective. IVIG prophylaxis has been believed notto prevent acquisition of the pathogen (see, Mandell et al., Eds.,Principles and Practice of Infectious Disease., 2nd ed., John Wiley &Sons, New York, 1985, pp. 37-43). This is a misconception because IVIGis usually given after infection is established. The applicants alsonote that even if IVIG were given before infection, effective levels ofimmunoglobulins would not be available to intercept pathogens at entrysites or portals such as on biomaterials, on burned or damaged tissues,and on mucosal surfaces, before or shortly after contamination becauseof dilution and deficient circulation.

The inconsistent benefit of immune globulin in preventing nosocomial andpost traumatic and burn infections may, in part, be explained by thevariable levels of antibodies in standard preparations, as well as therequired absence of IgA and IgM from IVIG preparations to prevent sideeffects. In 1992, a New England Journal of Medicine report stated,"Little is known about the variability in levels of antibodies to morecommon nosocomial pathogens, such as coagulase-negative staphyloccoci,or about the nature of the antibodies that confer protection, or evenwhether antibodies have a role in preventing infections associated withindwelling devices" (see, Siber, New Eng. J. Med., 327(4):269-271(1992)).

This invention solves the short-comings of IVIGs in preventing andtreating infections secondary to trauma, burns, surgery, andbiomaterials by applying a full repertoire immunoglobulin composition totissue surfaces and biomaterials to prevent microbial adhesion andcolonization (the acquisition of pathogens) and to pre-opsonize microbesin-situ for enhanced phagocytosis and killing. By preventing adhesion tosurfaces and by opsonizing bacteria on arrival and shortly afterward,bacteria are identified, made vulnerable, and targeted for neutrophiland macrophage phagocytosis and killing while bacterial numbers are lowbefore they can reproduce, release toxins, destroy tissue and formprotective biofilms. This process also assists antibiotic strategies,since bacteria are more vulnerable before attachment to surfaces.

The use of applied coating concentrates of globulins to tissue, mucosaland biomaterial surfaces allows high dosages of IgA and IgM, in additionto IgG, to be delivered directly to a wound. Currently available IVIGpreparations have IgA and IgM selectively removed to preventanaphylactoid reactions. Anaphylactoid reactions are not a danger whenan immune globulin composition is used locally at a wound or burn site.Including IgA in a creme, ointment, or lavage fluid that will be appliedto a wound or biomaterial is advantageous since IgA is known to blockadhesion of bacteria and to neutralize viruses. IgM enriched IVIGpreparations have been reported to be highly effective against gramnegative bacteria and endotoxins (see, Behre et al., AntimicrobialAgents and Chemotherapy, October 1992, pp.2139-2146); therefore,including elevated concentrations of IgM in a creme, ointment or lavagefluid is preferred. Macrophages and complement are naturally mobilizedand concentrated at wound sites and are available to respond to thebacteria opsonized by the therapeutically delivered polyvalent globulinsof the inventive compositions. The generation of immune complexes andinflammatory mediators, as occurs with high doses of IV preparations, isdiminished or prevented by local delivery. Equine or other animalderived plasma products, if utilized for human or animal therapy, arealso less likely to cause side effects by this method.

In summary, wound or biomaterial surface pretreatment, at time ofsurgery or shortly after trauma, allows the effective use of a fullrepertoire of globulins including IgG, IgM, and IgA at highconcentrations without side effects, before infection starts.

The immunoglobulin preparations of the present invention can be preparedby a number of methods. It is contemplated that an ideal method forobtaining the immunoglobulin preparations is to first obtain theimmunoglobulin fraction (cold ethanol fractionation process) from thesera from a large number of human donors. As needed, the immunoglobulinpool will be fortified with hyperimmune immunoglobulins obtained fromimmunized donors or donors with high antibody titers for specifiedbacteria or viruses. In addition, monclonal antibodies for specifiedbacteria and viruses can be added to the compositions.

In this invention, the full repertoire of immunoglobulin classes, IgG,IgA, IgM, is preferably used in the ointments, cremes, lavage fluids,etc.; however, the ointments, cremes, and lavage fluids could containonly IgG if desired. Preferably, the immunoglobulins will beconcentrated for high dosages. The immunoglobulins will constitute0.1-20 percent by weight of the ointment, creme, lavage fluid, etc.,with higher concentrations preferred (e.g., 10-20 percent by weight). Ifmonoclonal antibodies specific for certain microrganisms are added tothe immunoglobulin compositions, they will typically be present at0.01-1 percent by weight. The ointments, cremes, or lavage fluids willbe used locally by direct application to a wound or burn, or as a washor coating for a biomaterial device or implant (e.g., catheter). Inaddition, the compositions can be impregnated in or immobilized on amatrix carrier (e.g., fibrin, collagen, etc.) for sustained release orelution therefrom. The matrix carrier can be in the form of a wounddressing or other material placed in-situ at a wound or can be coated onthe body contacting surfaces of a biomaterial implant or device(catheter, etc.). The immunoglobulin compositions will ordinarily beprovided to patients at 2-100 mg/kg body weight; however, variation fromthis dose range can occur. The size of the wound or biomaterial implantcan dictate that smaller or larger quantities of the compositions beused.

Table 5 lists the concentration ranges and mean values forimmunoglobulins found in normal sera, as well as the proposedconcentrations of immunoglobulins to be used in wash lavage or washfluid preparations contemplated by this invention.

                  TABLE 5                                                         ______________________________________                                        Range of concentrations of immunoglobulins in                                 normal human sera in mg/dl as compared to the                                 concentrations used in lavage fluid preparations of                           the present invention                                                         Immunoglobulin                                                                           Normal Serum Level                                                                           Mean    Lavage                                      ______________________________________                                        IgG        600-1600       989      500-2000                                   IgM        45-150         100     100-300                                     IgA        60-330         200     100-500                                     ______________________________________                                    

As discussed above, compositions with elevated levels of IgM and IgA(200-300 mg/dl and 400-500 mg/dl, respectively) would provide benefitsin blocking adhesion of bacteria to biomaterials and certain tissues,which will prevent microbial pathogen adherence and colonization as wellas have enhanced activity towards gram negative bacteria and endotoxins.In addition, concentrated levels of IgA provide enhanced neutralizationof viruses and prevent viruses from infecting cells lining themucocutanous surfaces of the body. Since the broad spectrumimmunoglobulin compositions are being locally delivered, anaphylactoidreactions are avoided. Furthermore, side effects associated with IVIG(IgG only) preparations such as increased pulse rate and blood pressureare avoided by local delivery, thereby allowing elevated concentrationsof IgG (1700-2000 mg/dl or higher) to be administered to a patient.Concentrated levels of immunoglobulins (IgG, IgM, and IgA) enhance thein-situ pre-opsonization strategy contemplated by this invention. Thelavage fluids of the present invention will ordinarily be diluted insaline at neutral pH and will include stabilizing agents such as glucose(up to 20 mg/ml), polyethylene glycol (up to 2 mg/ml), glycine (up to0.3M), and albumin (preferably human up to 3 mg/ml). Buffer agents(e.g., acetate) could be included in the lavage fluids. Other basefluids (ethanol, etc.) and stabilizing agents (maltose, etc.), and thelike may also be used for the lavage fluids of the present invention.The lavage fluids of the present invention could be used as wash for allroutine surgeries including fiberoptic procedures, will have vaginal andgenitourinary applications, and can be used as a peritoneal wash orcombined with continuous peritoneal dialysate solutions.

Table 6 lists the concentration ranges of immunoglobulins in cremes,syrups, or other special viscous carriers (including lozenges andsuppositories), contemplated by this invention.

                  TABLE 6                                                         ______________________________________                                        Range of concentrations of immunoglobulins in mg/dl                           in a viscous carrier (creme, ointment, syrup) of                              the present invention.                                                        Immunoglobulin Class                                                                              Composition Level                                         ______________________________________                                        IgG                 2,500-20,000 mg/dl                                        IgM                 500-3,000 mg/dl                                           IgA                 500-5,000 mg/dl                                           ______________________________________                                    

Cremes, ointments, syrups, and the like, which are applied to thesurfaces of biomaterial devices and implants (catheters, etc.), or tothe surfaces of skin and of bandages and other dressings, as well asburned or damaged tissue provide an ideal mechanism for maintainingimmunoglobulins in-situ for extended periods of time. Because thecarrier is a lotion, syrup, oil, or thickening agent, theimmunoglobulins can be concentrated to levels 5-10 times greater thanthat used for lavage or wash fluids. As discussed above, stabilizers andother agents will be combined with the creme, ointment, syrup, coughdrops, etc. Sprays, syrups, and cough drops containing the fullrepertoire immunoglobulin compositions are an ideal method forrespiratory infection prevention and for delivery in times of epidemicrisk.

The immunoglobulin preparations to be used in this invention will betested for opsonic activity, viral neutralizing activity, andbactericidal activity with the addition of the complement system invitro to evaluate and standardize the potency of the preparations. Whenactivities are suboptimal, the preparations will be either fortifiedwith hyperimmune globulins or monoclonal antibodies to provide thenecessary antibody spectrum and level to cover the microbial strainspecificities required for effective prophylaxis and/or treatment. Table7 lists the major candidates for prophylaxis and treatment of wound,burn, nosocomial, and oral and respiratory infections of all types(including implanted devices).

                  TABLE 7                                                         ______________________________________                                                            Specific                                                                      Antibodies Estimated                                      Microorganism       Effective Concentration                                   ______________________________________                                        Staphylococcus aureus                                                                             1-50 μg/ml                                             S. epidermidis      1-50 μg/ml                                             Coagulase Neg. Staph.                                                                             1-50 μg/ml                                             Streptococcus (Groups A, B, and D)                                                                1-50 μg/ml                                             Pseudomonas aeruginosa                                                                            1-50 μg/ml                                             Escherichia coli    1-50 μg/ml                                             Enterobacter spp.   1-50 μg/ml                                             Klebsiella pneumoniae                                                                             1-50 μg/ml                                             Streptococcus pneumoniae                                                                          1-50 μg/ml                                             S. mutans           1-50 μg/ml                                             Hemophilus influenzae                                                                             1-50 μg/ml                                             Proteus spp.        1-50 μg/ml                                             Bacteroides gingivalis                                                                            1-50 μg/ml                                             Streptococcus pyogenes (Group A)                                                                  1-50 μg/ml                                             Mycoplasma pneumoniae                                                                             1-50 μg/ml                                             Respiratory Syncytial Virus                                                                       1-50 μg/ml                                             Influenza virus (A, B, and C)                                                                     1-50 μg/ml                                             Rhinovirus          1-50 μg/ml                                             ______________________________________                                    

An immunoglobulin composition of this invention which could be useduniversally in the treatment and prophylaxis of wounds, burns,nosocomial infections, and oral and respiratory infections would havespecific antibodies against each of the groups of potential pathogens ofTable 7 within the above concentration ranges. In particularapplications, the antibody titers for specific pathogens in theimmunoglobulin compositions can be five to twenty times greater thanthose specified in Table 7 (e.g., 5-1000 μg/ml). Compositions containinglower or higher antibody titers to less or more than the above listedpathogens might also provide protection from infections. For instance, apreparation containing high titer levels for S. aureus and P. auruginosamay provide acceptable results. However, it should be understood thatwound, burn, and nosocomial infections, etc., are commonly polymicrobialand the result of a wide variety of pathogens, therefore, hyperimmuneimmunoglobulin compositions should contain high titers of antibodies forat least two and preferably three, four, or five, or more, of thepathogens listed in Table 7. Note that Table 4 above demonstrates thathyperimmune immunoglobulin compositions raised against a pool ofinfectious pathogens provides the optimum protection. Therefore, thisinvention contemplates a "polyclonal cocktail" of antibodies specificfor key pathogens that normally gain entrance to all wounds, etc., asneeded and determined by in vitro functional assays.

The invention also contemplates the use of monoclonal cocktails preparedagainst specific epitopes on the immunogenic antigens from the pathogensin Table 7. In this case, the effective concentrations would be 1-2orders of magnitude lower than those indicated in Table 7. Specifically,concentrations of 0.01-5 μg/ml of monoclonal antibodies would be presentin the compositions.

Furthermore, the invention also contemplates supplementingimmunoglobulin compositions with monoclonal antibodies specific for therelevant pathogens as needed. As discussed above, the concentration ofthe monoclonal antibodies added to the compositions would be in the0.01-5 μg/ml range.

Immunoglobulin polyclonal cocktail preparations, monoclonal cocktails,and immunoglobulin preparations supplemented with monoclonal antibodies,can be prepared for specific applications to combat the major pathogensassociated with those applications. The immunoglobulins for specificpathogens would preferably have an effective concentration of 1-50 μg/mlof antibodies for those pathogens, and could have higher effectiveconcentrations (e.g., 5-1000 μg/ml) as described above. In addition, ifthe compositions contained monoclonal antibodies specific for thepathogens associated with a particular application, they would bepresent at a concentration of 0.01-5 μg/ml. The compositions willcontain those antibodies which are against the most clinically relevantstrains or types of organisms.

The major pathogens to defend against will vary depending on the site ofinfection. For example, a contact lens wash solution should includeimmunoglobulins with antibodies or monoclonal antibodies specific for S.epidermidis and P. aeruginosa. In genitourinary catheter applications,the compositions should include immunoglobulins with antibodies ormonoclonal antibodies specific for at least two of the followingmicroorganisms: E. coli, Enterobacter spp., Proteus spp., and P.aeruginosa. In intravenous, intraarterial, or intraperitoneal catheterapplications, the compositions should include immunoglobulins withantibodies or monoclonal antibodies specific for at least two of thefollowing microorganisms: S. aureus, P. aeruginosa, E. coli, and S.epidermidis. Compositions to be used with wound (surgical or otherwise)and burn dressings should include immunoglobulins with antibodies ormonoclonal antibodies specific for at least two of the followingmicroorganisms: S. aureus, Enterobacter spp., S. epidermidis, and P.aeruginosa. Compositions to be used in combination with biomaterialimplants and devices (catheters, artificial hearts, etc.), shouldinclude immunoglobulins with antibodies or monoclonal antibodiesspecific for at least two of the following pathogens: S. epidermidis, S.aureus, E. coli, Enterobacter spp., or P. aeruginosa. Oral compositions(lozenges, syrups, etc.) should include immunoglobulins with antibodiesor monoclonal antibodies specific for at least two of the followingpathogens: S. aureus, S. mutans, and Bacteroides gingivalis.Compositions used for oral, nasopharyngeal, and respiratory infections(e.g., aerosol and non-aerosol sprays, lozenges, syrups, etc.) shouldinclude immunoglobulins with antibodies or monoclonal antibodiesspecific for at least two of the following pathogens: Streptococcusmutans, B. gingivalis, S. pyogenes (group A), S. pneumoniae, K.pneumoniae, P. aeruginosa, S. aureus, M. pneumoniae, H. influenzae,Respiratory Syncytial Virus, Influenza Virus (A, B, and C), andrhinoviruses.

The time of application of the full repertoire immunoglobulincompositions is important. Within six hours after a surgical wound orburn site occurence, or after cleaning a wound or burn, a biofilm isformed over the site which includes bacteria and viral agents. Thebiofilm can shield the microbial agents against antibiotics, intravenousimmunoglobulins, and phagocytes; therefore, the biofilm acts arepository for pathogens which cause chronic and recurrent infections.By applying the full repertoire broad spectrum immunoglobulincomposition at the wound or burn site immediately after cleaning orsurgery and prior to bacterial attachment or biofilm formation preventsadhesion of the bacteria and pre-opsonizes the bacteria for phagocytickilling and removal before toxin release.

The protective activity of the full repertoire broad spectrumimmunoglobulin composition could be enhanced by providing antibiotics,antivirals, antiinflammatory and healing compounds in combination withthe immunoglobulins. For example, biocides, surfactants, bacterialblocking receptor analogues, cytokines, growth factors, macrophagechemotactic agents, cyphalosporins, aminoglycosides, fluoroquinolones,etc., could be provided at therapeutically acceptable levels in thelavage fluids, sprays (both aerosol and non-aerosol), ointments, cremes,syrups, lozenges, suppositories, and the like, of the present invention.

The full repertoire broad spectrum immunoglobulin compositions mayideally be immobilized within fibrin, collagen, gelatin, hyaluronan(hyaluronic acid), polysacharide, or other biocompatible orbiodegradable materials that are to be placed in-situ at a wound or burnsite. This would insure that antibodies to particular pathogens remainpresent throughout the healing process. The antibodies of theimmunoglobulin compositions could ideally have a slow, sustained releaseor elution from the matrix materials. Ideally, the layers of the matrixmaterials with immobilized immunoglobulins would be biodegradable.Antibiotic, antiviral, antiinflammatory and healing compounds wouldideally be used in combination with the immunoglobulin composition, andthese compounds would be impregnated into the biocompatible material.Catheters, ventilators, and implantable devices such as vascular graftsand total joints would ideally have the full repertoire broad spectrumimmunoglobulin compositions of the present invention, as well asantibiotic and antiviral compounds, immobilized on an external orinternal, body or blood contacting surface. Implantable devices arefrequently responsible for severe infections; therefore, thecompositions of this invention would have immediate application incombination with these devices.

It should be understood that the hyperimmune globulin compositions ofthe present invention have veterinary applications as well as humanhealth care utility.

While the invention has been described in terms of its preferredembodiments, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims.

Having thus described our invention, what we claim as new and desire tosecure by Letters Patent is as follows:
 1. A biomaterial, comprising:adevice insertable into a human or animal host's body at a surgical ortrauma site of a wound, burn or biomaterial implant site, said devicehaving a body contacting surface; and an immunoglobulin compositioncoated on said surface of said device, said immunoglobulin compositioncomprising a combination of IgG, IgM and IgA, wherein said compositioncontains elevated levels of IgM between 200-300 mg/dl and IgA between400-500 mg/dl when compared with normal serum levels, wherein saidimmunoglobulin composition is specific for microorganisms.
 2. Thebiomaterial of claim 1, wherein said immunoglobulin compositioncomprises monoclonal antibodies.
 3. The biomaterial of claim 1 whereinsaid device is selected from the group consisting of contact lenses,catheters, ventilators, vascular grafts, internal fixation devices, andjoints.
 4. The biomaterial of claim 1 wherein said immunoglobulincomposition is immobilized on said surface of said device.
 5. Thebiomaterial of claim 1 further comprising a matrix carrier on saidsurface of said device wherein said matrix carrier is selected from thegroup consisting of fibrin, collagen, gelatin, polysaccharides, andhyaluronan.
 6. The biomaterial of claim 1, wherein said immunoglobulincomposition is specific for at least two microorganisms selected fromthe group consisting of Staphylococcus aureus, Staphylococcusepidermidis, Pseudomonas aeruginosa, Escherichia coli, Enterobacterspp., Streptococcus mutans, Bacteroides gingivalis, Streptococcus(Groups A, B and D), Coagulase Negative Staphylococci, Klebsiellapneumoniae, Hemophilus influenzae, Proteus spp., Mycoplasma pneumoniae,Streptococcus pyogenes, Respiratory Syncytial Virus, Influenza Virus (A,B, and C), and rhinoviruses.
 7. The biomaterial of claim 6, wherein saidimmunoglobulin composition comprises monoclonal antibodies.